Transferrin Receptor-Mediated Cellular Uptake of Fluorinated Chlorido[N,N′-bis(salicylidene)-1,2-phenylenediamine]iron(III) Complexes

Fluorinated chlorido[salophene]iron(III) complexes (salophene = N,N′-bis(salicylidene)-1,2-phenylenediamine) are promising anticancer agents. Apoptosis and necrosis induction have already been described as part of their mode of action. However, the involvement of ferroptosis in cell death induction, as confirmed for other chlorido[salophene]iron(III) complexes, has not yet been investigated. Furthermore, the mechanism of cellular uptake of these compounds is unknown. Therefore, the biological activity of the fluorescent chlorido[salophene]iron(III) complexes with a fluorine substituent at positions 3, 4, 5, or 6 at the salicylidene moieties (C1–C4) was evaluated in malignant and nonmalignant cell lines with focus on the involvement of the transferrin receptor-1 (TfR-1) in cellular uptake, the influence of the complexes on mitochondrial function, and the analysis of the molecular mechanism of cell death. All complexes significantly decreased the metabolic activity in the tested ovarian cancer (A2780, A2780cis), breast cancer (MDA-MB 231), and leukemia (HL-60) cell lines, while the nonmalignant human stroma cell line HS-5 at a concentration of 0.5 μM, which represents the IC50 of the complexes in most of the used tumorigenic cell lines, was not affected. The mitochondrial function was impaired, as evidenced by a reduced mitochondrial membrane potential ΔΨm and decreased mitochondrial activity. Besides apoptosis and necroptosis, ferroptosis was identified as part of the mode of action. It was further demonstrated for the first time that fluorinated chlorido[salophene]iron(III) complexes downregulate TfR-1 expression, comparable to ferristatin II, an iron transport inhibitor that acts via TfR-1 degradation. FerroOrange staining further indicated that the complexes strongly increased the intracellular iron(II) level as a driving force to induce ferroptosis. In conclusion, these fluorinated chlorido[salophene]iron(III) complexes are potent, tumor cell-specific chemotherapeutic agents, with the potential to treat various types of cancers.

Table S2: Percentage of TfR-1/-Actin expression in MDA-MB 231 cells after 4 h treatment with Ferristatin II at concentrations ranging from 0.5 µM to 100 µM, respectively.The TfR-1/-Actin expression of untreated cells was set at 100%.

Figure S31 :
Figure S31: Metabolic activity of HL-60 cells treated with the complexes C1 -C4 at concentrations of 0.1 µM (filled), 0.5 µM (horizontally striped), 1 µM (striped across) and 10 µM (dotted) for 72 h.Cisplatin and SP at a concentration of 1 µM served as references.Metabolic activity in the absence of the complexes was set at 100%.Data are expressed as mean + SE of five independent experiments.The asterisks (* p<0.05, ** p<0.005 and *** p<0.0005 against the cells without addition of compound) represent statistical significance.

Figure S32 :
Figure S32: Metabolic activity of A2780 cells treated with the complexes C1 -C4 at concentrations of 0.1 µM (filled), 0.5 µM (horizontally striped), 1 µM (striped across) and 10 µM (dotted) for 72 h.Cisplatin and SP at a concentration of 1 µM served as references.Metabolic activity in the absence of the complexes was set at 100%.Data are expressed as mean + SE of five independent experiments.The asterisks (* p<0.05, ** p<0.005 and *** p<0.0005 against the cells without addition of compound) represent statistical significance.

Figure S33 :
Figure S33: Metabolic activity of A2780cis cells treated with the complexes C1 -C4 at concentrations of 0.1 µM (filled), 0.5 µM (horizontally striped), 1 µM (striped across) and 10 µM (dotted) for 72 h.Cisplatin and SP at a concentration of 1 µM served as references.Metabolic activity in the absence of the complexes was set at 100%.Data are expressed as mean + SE of five independent experiments.The asterisks (* p<0.05, ** p<0.005 and *** p<0.0005 against the cells without addition of compound) represent statistical significance.

Figure S34 :Figure S35 :
Figure S34: Metabolic activity of HS-5 cells treated with C1 -C4 (0.5 µM, 1 µM and 10 µM) for 72 h.Metabolic activity in the absence of the complexes was set at 100%.Data are expressed as mean + SE of six independent experiments.The asterisks (* p<0.05 and ** p<0.005 against the cells without addition of compound) represent statistical significance.

Figure S38 :
Figure S38: Western Blot analysis of the TfR-1 content in MDA-MB 231 cells in the absence (control; lane 2) and the presence of Ferristatin II at the concentrations of 0.5 µM, 1 µM, 10 µM, 50 µM and 100 µM (lanes 3-7) after incubation for 4 h.The biotinylated molecular weight ladder (Biot.Ladder) is shown as lane 1. β-Actin was used as loading control.